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Zagazig J. Agric. Res., Vol. 46 No. (6A) 2019

BIOSYNTHESIS OF ZINC NANOPARTICLES USING CULTURE

FILTRATES OF Aspergillus, Fusarium AND Penicillium FUNGAL SPECIES

AND THEIR ANTIBACTERIAL PROPERTIES AGAINST GRAM-POSITIVE

AND GRAM-NEGATIVE BACTERIA

Mona E. Hefny*, Fatma I. El-Zamek, H.I. Abd El-Fattah and S.A.M. Mahgoub

Agric. Microbiol. Dept., Fac. Agric., Zagazig Univ., Egypt

Received: 04/08/2019 ; Accepted: 27/08/2019

ABSTRACT: The present study demonstrates a positive correlation between zinc metal tolerance

ability of an isolated fungi and their potential for the synthesis of zinc oxide nanoparticles (ZnO-NPs).

A total of 5 fungal cultures were isolated from the rhizospheric soils of plants naturally growing at

Sharkia Governorate in Egypt and identified based on morphological characteristics. These isolates are

belongs to Aspergillus niger (An), Aspergillus tubulin (At), Aspergillus fumigatus(Af), Penicillium

citrinum (Pc) and Fusarium oxysporum (Fo). These isolates were used in the synthesis of zinc-oxide

nanoparticles (An-ZnO-NPs, At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-NPs and Fo- ZnO-NPs) using Zinc

sulfate as the precursor compared to the references strains of A. tubingensis Mosseray AUMC

No.6915, A. fumigatus Fresenius AUMC No.48 and A. terreus Thom AUMC No.75. Aspergillus and

Fusarium isolates have been shown to have a high zinc metal tolerance ability and a potential for

extracellular synthesis of ZnO nanoparticles under ambient conditions. The synthesized ZnO-NPs

were tested by the detection of a notable absorption peak at 285 to 296 nm, appearing in UV–Vis

spectra due to surface-plasmon-resonance. Transmission electron microscope (TEM) results revealed

that An-ZnO-NPs, At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-NPs and Fo- ZnO-NPs exhibited a crystalline

structure with hexagonal wurtzite shape (30–100 nm size). ZnO nanoparticles exhibited excellent

antibacterial activity against tested Gram-positive and Gram-negative bacteria. The ZnO nanoparticles

showed better antibacterial activity against Staphylococcus. aureus, Listeria. monocytogenes and

Bacillus cereus compared to Salmonella enterica, Escherichia coli and Pseudomonas aerogenosa. The

effectiveness of inhibition of the microbial biofilms formation of S.aureus, L. monocytogenes and

B. cereus compared to S. enterica, E. coli and P. aerogenosa was analyzed at a concentration of 100 μg/ ml.

Key words: Fusarium oxysporum, Aspergillus fumigatus, Zinc sulfate, ZnO nanoparticle

characterization, antimicrobial activity; bacterial pathogens.

INTRODUCTION

Synthesis of nanoparticles employing

microorganisms has attracted much due to their

usual optical, chemical, photoelectron chemical

and electronic properties and many biological

organisms, such as bacteria, fungus, yeasts and

plants either intra or extracellular (Castro-

Longoria et al., 2010). Eukaryotic organisms

such as fungi are extremely good candidate for

the synthesis of metal nanoparticles. The fungi

give nanoparticles with good monodispersity

and well-defined dimensions. Because of their

tolerance and metal bioaccumulation ability,

fungi are taking the center stage of studies on

biological generation of metallic nanoparticles

(Sastry et al., 2003). A distinct advantage of

using fungi in nanoparticle synthesis is the ease

in their scale-up (e.g., using a thin solid

substrate fermentation method). Given that fungi

are extremely efficient secretors of extracellular

enzymes, it is thus possible to easily obtain

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Biotechnology Research

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E-mail address: [email protected]

Hefny, et al.

large-scale production of enzymes. Further

advantages of using a fungal-mediated green

approach for synthesis of metallic nanoparticles

include economic viability and ease in handling

biomass. However, a significant drawback of

using these bio-entities in nanoparticles synthesis

is that the genetic manipulation of eukaryotic

organisms as a means of overexpressing specific

enzymes (e.g., the ones identified in synthesis of

metallic nanoparticles) is relatively much more

difficult than that in prokaryotes. The biological

method of the ZnO NPs synthesis is gaining

importance due to its simplicity, eco-friendliness

and extensive antimicrobial activity (Gunalan et

al., 2012). The main factor for the increase of

the resistance pathogenic bacteria is overuse of

antibiotics and this has led to the emergence and

spread of resistant pathogens and resistant genes

in them (van den Bogaard and Stobberingh,

2000). Nanoparticles as antimicrobial agents are

better efficiency on resistant bacteria, less

toxicity and heat resistance. Among metal oxide

Nanoparticles, ZnO have many significant

features such as chemical and physical stability,

high catalysis and effective antibacterial activity

(Kalyani et al., 2006). Recently, ZnO NPs have

been used in food packaging materials and

various matrices and methods for incorporation

of ZnO into those matrices have been reported.

ZnO is incorporated into the packaging matrix,

free to interact with the food materials offering

preservatory effects (Espitia et al., 2012).

Presently, ZnO NPs have found applications in

sunscreens, paints and coatings as they are

transparent to visible light and offer high UV

absorption (Franklin et al., 2007) and are also

being used as an ingredient in antibacterial

creams, ointments and lotions, self cleaning

glass, ceramics and deodorants (Li et al., 2008).

ZnO nanoparticles have been lately tested for

their antimicrobial potential and seem to possess

both antibacterial and antifungal potential. They

are active against both Gram-positive and Gram-

negative bacteria and also show considerable

activity against more resistant bacterial spores

(Azam et al., 2011). It was also observed that

doping of ZnO NPs with other metals such as

gold, silver, chromium etc. improved the

antimicrobial activity of ZnO NPs (Shah et al.,

2014; Jime´nez et al., 2015). Also, inhibitory

effects of ZnO nanosuspension are correlated

with their size and concentration, with smaller

particles offering better inhibitions in higher

concentrations (Buzea et al., 2007; Padmavathy

and Vijayaraghavan, 2008). ZnO NPs, in

particular, are environment friendly, offer easy

fabrication and are non-toxic, biosafe and

biocompatible making them an ideal candidate

for biological applications (Rosi and Mirkin,

2005; Mohammad et al., 2010). Additionally,

as per the US Food and Drug Administration,

zinc sulphate with other three zinc compounds

(Zinc Oxide, Zinc Nitrate, Zinc Choloride) have

been listed as generally recognized as safe

(GRAS) material (FDA, 2015). Various

chemical methods have been proposed for the

synthesis of ZnO NPs, such as reaction of zinc

with alcohol, vapor transport, hydrothermal

synthesis, precipitation method etc. However,

these methods suffer various disadvantages due

to the involvement of high temperature and

pressure conditions and the use of toxic

chemicals. The advantages of using inorganic

oxides nanoparticles as antimicrobial agents are

their greater effectiveness on resistant strains of

microbial pathogens, less toxicity and heat

resistance (Zhang et al., 2010). ZnO has

biocidal action and strong antibacterial agent

due to its physiochemical properties and

biocompatibility (Mirzaei and Darroudi,

2017). Plus, the crystallite size and the nanoparticle

shape have an effect on the antibacterial activity

which smaller ZnO nanoparticles have higher

antibacterial activity. Several reports have

addressed the harmful impact of nanomaterials

on living cells, but relatively low concentrations

of ZnO are nontoxic to eukaryotic cells (Zaveri,

et al., 2010; Krishna et al., 2011) stated that

ZnO nanoparticles significantly inhibit the

growth of a wide range of pathogenic bacteria

under normal visible lighting condition. Thus,

the current study aims to isolate fungi from soil

contaminated with heavy metals for

biosynthesizing of zinc-oxide nanoparticles using

zinc sulfite as the precursor compared to the

references strains of A. tubingensis Mosseray

AUMC No.6915, A. fumigatus Fresenius

AUMC No.48 and A. terreus Thom AUMC

No.75. The second aim of this study was to

evaluate the zinc-oxide nanoparticles efficiency


against Gram-positive and Gram-negative bacteria

as antibacterial agents.

MATERIALS AND METHODS

Preparation of Stock Solution of Zinc

Sulphate

The stock solution of zinc sulphate was

prepared by dissolving 1.61 g/l of zinc sulfate in

20 ml of sterilized distilled water to get the 10

mM concentration and then stored for use in the

experimentations.

Sampling and Isolation of Fungi

The soil samples (n=10) were collected from the rhizospheric soils of plants naturally growing at Sharkia Governorate, Egypt. These samples were used for isolation of metal-tolerant fungi. Potato Dextrose Agar (PDA: potato extract 1000 ml, sucrose 20 g and agar 15g) were used for isolation of fungi. This media was containing 1mM of zinc sulfate (Jain et al., 2013). Plates were incubated at 28°C for 72 hr. The tolerant colonies were picked up and transferred to Czapek Dox Agar slants and preserved at 4°C in a refrigerator.

Strains Used

Fungal strains of Aspergillus tubingensis

Mosseray AUMC No.6915, A. fumigatus

Fresenius AUMC No.48 and A. terreus Thom

AUMC No.75 were obtained from The Center

of Fungi Science, Assiut University, Egypt.

These standard of fungi strains were used as a

references strains in the current study. While

bacterial strains of Staphylococcus aureus

ATCC 6538, Listeria monocytogenes Scott A,

Salmonella enterica serovar Enteritidis PT4,

Pseudomonas aeruginosa and Escherichia coli

ATCC 8739 were obtained from Egyptian

Culture Collection (MERCIN), Ain Shams

University, Cairo, Egypt.

Metal Tolerance Profiles of Fungal Isolates

A maximum tolerable concentration (MTC)

assay was performed to determine the zinc metal

tolerance ability of fungal isolates according to

the method of Jain et al. (2013). The experimental

plates were prepared by supplementing PDA

medium with varying amounts of zinc sulfate to

obtain final concentrations of Zn+2 ions in the

ranges of 200, 400, 800, 1600, 2000, 2500, 3000

and 3200 μg ml−1. Plates without Zn+2 ions were

used as a control. Each plate was subdivided

into four equal sectors and an inoculum of test

fungi (106 fungal propagates ml−1) was spotted

on the media surface. After inoculation, the

plates were incubated at 28°C for 4 days under

dark conditions to examine the fungal growth.

The experiment was done in triplicate. The

maximum concentration of Zn+2 ions in the

medium which allowed the growth of a fungus

was taken as MTC.

Extracellular Synthesis of (ZnO-NPs)

Nanoparticles by Fungal Strains and

Isolates

Biomass was produced by cultivation of isolated and strains fungi in Malt Glucose Yeast Peptone (MGYP) broth composed of yeast extract and malt extract 0.3% each, glucose 1%, peptone 0.5%. Biomass preparation of Aspergillus niger, Aspergillus tubulin, Aspergillus fumigatus, Penicillium citrinum and Fusarium oxysporum used in this study have originally been isolated from soil contaminated with heavy metals compared to the standard strains of A. fumigatus, A.tubulin, Fusarium oxysporum and Penicillum citrinum. Fungi were grown in 250 ml Erlenmeyer flasks each containing 50 ml liquid medium containing (g/l): KH2PO4 7.0 g; K2HPO4 2.0 g; MgSO47H2O 0.1 g; (NH4)2SO4 1.0 g; yeast extract 1.0 g and glucose 15.0 g. Inoculated media were incubated at 28 ± 2 0C and 180 rpm for 5 days, after which each fungal biomass was separated using Whatman filter paper No. 1 and extensively washed by deionized water. The collected fungal biomass was transferred to 100 ml of deionized water in the 250 ml Erlenmeyer flask and further incubated at 140 rpm for 72 hr., in an orbital shaker. Each fungal biomass was filtered again with Whatman filter paper No. 2 and the collected cell-free filtrate was subjected to biosynthesis of zinc oxide nanoparticles (Jain et

al., 2011). The treatments have been named An-ZnO-NPs, At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-NPs and Fo- ZnO-NPs. 10 ml of 3.0 mM ZnSO4 salt were added to 10 ml filtrate of each fungus (1;1) and adjusting the pH to 6.5 and incubated in orbital shaker for 150 rpm at 32ºC for 72 hrs., in adark condition. White precipitate deposition at the bottom of the flask indicated the formation of nanoparticles. White aggregate

Hefny, et al.

formed at the bottom of the flask was separated from the filtrate by centrifugation at 10,000 rpm for 10 min. (Baskar et al., 2013). Simultaneously, a positive control and negative control were maintained by incubating the each fungus mycelium with de-ionized water and Zinc sulphate solution (Dhoble and Kulkarni

2015).

Characerization of ZnO-NPs

UV–Vis spectra analysis

Each sample (An-ZnO-NPs, At-ZnO-NPs,

Af-ZnO-NPs, Pc- ZnO-NPs and Fo- ZnO-NPs)

was measured for its maximum absorbance

using UV-Vis spectrophotometry (Jasco

Corporation, Tokyo, Japan). The optical

property of ZnO nanoparticles was analyzed via

ultraviolet and visible absorption spectroscopy

in the range of 200–800 nm. This techniques

were carried out at The Center of Fungi in

Faculty of Science, Al-Azhar University, Cairo,

Egypt.

Transmission electron microscopy (TEM)

TEM analysis was performed to determine

the morphology, size and shape of the ZnO

nanoparticles. TEM measurements were done by

HITACHI H-800, operating at 200 kV. The

TEM grid was prepared by placing a drop of the

bio-reduced diluted solution on a carbon-coated

copper grid and later drying it under a lamp. The

size distribution and stability of ZnO NPs were

registered. This techniques were carried out at

The Center of Fungi in Faculty of Science, Al-

Azher University, Cairo.

Antibacterial Assay

Inhibition Zone

The technique was performed using agar well

diffusion (Magaldi et al., 2004). Six different

pathogenic bacterial strains were used in the

current study. Gram positive bacteria i.e.

Bacillus cereus, Staphylococcus aureus and

Listeria monocytogenes as well as Gram

negative bacteria i.e. Escherichia coli,

Salmonella enterica and Pseudomonas

aeruginosa were used as test organisms and

initially cultured in nutrient broth. All the

pathogenic microbes were grown overnight in

Mueller-Hinton broth (MHB). 100 μl culture of

each strain was spread evenly on a Mueller-

Hinton agar (MHA) plate. Then the wells were

made into agar at 4 mm diameter using sterile

cork-borer with the distance between well and

another more than 22 mm. 100 μL of each An-

ZnO-NPs, At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-

NPs and Fo-ZnO-NPs was placed over the MHA

plates. The plates were incubated at 35°C for 24

hr. β-lactams group (AMP: ampicillin),

fluoroquinolones group (OFX: ofloxacin) and

aminoglycosides group (AK: amikacin) were

used as the control. The diameters of inhibition

zone against the tested bacteria (Yuvaraja et al.,

2017) were recorded in millimeter using metric

ruler.

Growth-reduction

To examine bacterial growth, overnight

cultures of approximately 1 × 109CFU/ml were

diluted 100-fold into 50 ml of Tryptone Soya

Broth in 250 ml flasks. 100 μl of each An-ZnO-

NPs, At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-NPs

and Fo- ZnO-NPs suspensions were added to the

respective flasks. Cultures were then grown for

up to 48 hr., at 200 rpm, 37°C. Bacterial growth

was measured by optical density at 600 nm

(OD600).

Antibiofilm formation by Microtiter Plate

(MTP) Method

All bacterial strains were grown overnight at

37°C as pure cultures on Tryptone Soya agar.

The single colonies were inoculated in 3 ml

Tryptone Soya Broth (TSB). Suspensions were

incubated for 24 hr., at 37°C and then diluted at

1 : 40 in a fresh TSB (2.4 × 107 CFU/ml) using

0.5 MacFarland standard tube. This dilution was

used as the inoculum in the microtiter plate test.

Microtiter plate test was performed according to

Dubravka et al. (2010) and Stepanović et al.

(2000). For each strain, 100 μl aliquots of

prepared suspension culture were inoculated into

four wells of the 96-well tissue culture plates

containing fresh TSB (Nunclon Delta, Nunc,

Roskilde, Denmark), then each treatment was

received 100 μl of ZnO nanoparticles. Each

culture plate included a negative control, four

wells with TSB were used. The plates were

incubated at 37°C for 24 hr. Afterwards, content

of each well was removed by aspiration and the

wells were rinsed three times with 250 μl sterile

physiological saline. The plates were dried in

inverted position. The attached bacteria were


fixed for 15 minutes at room temperature by

adding 200 μl volumes of methanol into each

well. The plates were stained with 200 μl

aqueous solution of crystal violet 0.5% (Crystal

Violet, Fluka) for 15 minutes at room

temperature. Following staining, the plates were

rinsed under running water until there was no

visible trace of stain. The stain bound to bacteria

was dissolved by adding 200 μl of 95% ethanol.

Then microplates were washed, air dried and

adherent biofilm was dissolved in 33% (V/V)

glacial acetic acid. To determine the potential of

bacterial pathogens treated with An-ZnO-NPs,

At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-NPs and

Fo- ZnO-NPs to form biofilm, the microtiter

plate-based assay and the biofilm formation on

abiotic surfaces was quantified as described by

Stepanović et al. (2007). Biofilm formation was

measured at 570 nm in an ELISA reader set

(Thermo Scientific Multiskan FC, USA), and

expressed in optical density (OD) values. The

mean OD value of negative controls (ODNC) was

0.070 ± 0.005. The treatments were considered

biofilm producers when their OD values were

three times greater than the standard deviation of

the mean ODNC . Additionally, the bacterial

strains with An-ZnO-NPs, At-ZnO-NPs, Af-

ZnO-NPs, Pc- ZnO-NPs and Fo- ZnO-NPs

showing ability to produce biofilm were

classified as weak (ODNC < OD ≤ 2×ODNC),

moderate (2×ODNC < OD ≤4×ODNC) or strong

(OD >4×ODNC biofilm producers (Li et al.,

2012). Biofilm production by each strain was

determined from four independent experiments

and each independent experiment included

triplicates for each bacterial strain. The mean of

the twelve absorbance values of each isolate was

recorded as a result. Absorbance values > ± 4 ×

the standard deviation were rejected.

Statistical Analysis

Data from microbiological analyses were

entered into Excel 2010 and calculated for

standard division (±SD) for all experiments.

RESULTS AND DISCUSSION

Identification of Fungal Isolates

Preliminary identification of fungi was performed on the basis of morphological parameters such as color, spore shape,

arrangement, and hyphal branching pattern after staining with cotton blue. Identification of fungi is still heavily dependent upon microscopic observation of the physical appearance of both spores and spore-bearing structures, usually achieved by first obtaining the fungi in pure culture. Examination of just the mycelium rarely enables an identification, nor are spores alone sufficient as there are many species producing very similar spores. The UK has an organised network, the UK National Culture Collection (www.ukncc.co.uk), through which it is possible to obtain appropriate taxonomic advice and services on many microorganisms, including fungi. Also, the further identifications were carried out in the Department of Plant Pathology, Faculty of Agriculture, Zagazig University, Zagazig, Egypt compared to the references strains of fungi. All the isolates were belong to Aspergillus niger, Aspergillus tubulin, Aspergillus fumigatus, Penicillium citrinum, and Fusarium oxysporum. All these isolates were used in biosynthesis of zinc oxide nanoparticles.

Metal Tolerance Profile of Fungal Isolates

All the five fungal isolates were subjected to

a screening for metal tolerance towards zinc,

and the results were expressed in terms of MTC.

A higher proportion (100%) of fungal isolates

showed significant tolerance with a varying

degree of magnitude. The genus Aspergillus

exhibited a more prominent level of zinc metal

tolerance followed by Fuzarium then

Penicillium. It is evident from the results that A.

niger, A. tubulin, A. fumigatus, F. oxysporum

and P. citrinum isolates have tremendous zinc

metal tolerances with a MTC value of 3000 and

2500 μg ml−1, respectively (Table 1). Due to its

maximum MTC value, A. niger, A. tubulin, A.

fumigatus, P. citrinum and F. oxysporum

isolates were selected for further studies on

extracellular synthesis of ZnO nanoparticles

compared to the references strains. The current

study approaches to establish the relationship

between metal tolerance ability of a soil fungus

and its potential for synthesis of ZnO

nanoparticles. Jain et al. (2013) found that

Aspergillus aeneus isolate NJP12 has been

shown to have a high zinc metal tolerance

ability and a potential for extracellular synthesis

of ZnO nanoparticles under ambient conditions.

Hefny, et al.

Table 1. Zinc metal tolerance profile for ZnSO4 of fungal isolates after 96 hr.

Isolate A maximum tolerable concentrations (MTC) (μg ml−1

)

200 400 800 1600 2000 2500 3000 3200

Aspergillus niger +++ +++ +++ +++ +++ +++ ++ -

Aspergillus tubulin +++ +++ +++ +++ +++ +++ ++ -

Aspergillus fumigatus +++ +++ +++ +++ +++ +++ ++ -

Fusarium oxysporum +++ +++ +++ +++ +++ ++ - -

Penicillium citrinum +++ +++ +++ ++ + + - -

Extracellular Biosynthesis of Zinc Oxide

Nanoparticles and Physical Characteristics

The ZnO nanoparticles were prepared in accordance with the methods described above. The extracellular synthesis of ZnO nanoparticles was carried out by exposure of a precursor salt solution (ZnSO4, ZnO, Zn(NO3)2, ZnCl2) (Zn+2 ions; 3.0 mM) to fungal cell-free filtrate obtained by incubating the fungus in an aqueous solution. The viability experiment results showed that fungal cells were viable till the end of reaction (72 hr.). During the synthesis process of ZnO nanoparticles, the initially colorless solution will turn to the milky solution (Fig. 2). This has indicated that the ZnO nanoparticles were successfully produced by cell-free filtrate. Jain et al. (2013) found that the synthesis of ZnO spherical nanoparticles coated with protein molecules by A. aeneus which served as stabilizing agents. Also, they showed that the role of fungal extracellular proteins in the synthesis of nanoparticles indicated that the process is nonenzymatic but involves amino acids present in the protein chains. Several authors showed that A. fumigatus (Rajan et al.,

2016), A. niger (Kalpana et al., 2018), F. oxysporum (Ahmed et al., 2003) and P. citrinum (Honary et al., 2013) have ability to

synthesis nanoparticles from metal salts. The synthesized ZnO nanoparticles from tested fungi are presented in Fig. 1. All ZnO nanoparticles have shown a strong absorption peak at around 285 to 296 nm. Rajendran and Sivalingam

(2013) found that ZnO nanoparticles have shown a strong absorption peak at around 360 nm. TEM results revealed that An-ZnO-NPs, At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-NPs and Fo- ZnO-NPs exhibited a crystalline structure

with hexagonal wurtzite shape (30–100 nm size) (Fig. 2).

Evaluation of Antibacterial Activity

Inhibition Zone method

The results of inhibition zones of ZnO nanoparticles compared to three tested antibiotic compounds against B. cereus, L. monocytogenes, S. aureus, E. coli, S. enterica and P. aerogenosa are presented in Table 2. The larger zone was observed at B. cereus and S. aureus culture which is 14 to 26 mm compared to the L. monocytogenes culture with 17 to 25 mm of inhibition zone with all tested fungi except Penicillium citrinum which is 12 to 15 mm. While the inhibition zone by the antibiotics were observed at B. cereus, S. aureus and L. monocytogenes culture which is 20 to 30 mm compared to the E. coli, S. enterica and P. aerogenosa cultures with 7 to 27 mm. On the other hand, the inhibition zones of ZnO nanoparticles were observed at E. coli, S. enterica and P. aerogenosa cultures which is 11 to 18mm with all tested fungi. Based on the results obtained, it can be suggested that Gram-negative bacteria are more resistance to ZnO nanoparticles compared to Gram-positive bacteria. Also, ZnO nanoparticles was more effective against Gram negative compared to the antibiotics especially when compared to the antibiotic (AMP). This supports by Premanathan et al.

(2011) who reports that ZnO nanoparticles showed a much stronger antibacterial effect on Gram-positive bacteria than on Gram-negative ones. Stoimenov et al. (2002) and Fu et al.

(2005) elucidated by the possibilities of membrane damage caused by electrostatic interaction between ZnO and cell surface.


Fig. 1. UV-visible spectrum of ZnO nanoparticles suspension biosynthesized by Aspergillus niger

(An), Aspergillus tubulin (At), Aspergillus fumigatus (Af) and Fusarium oxysporum (Fo).

Fig. 2. Image of ZnO nanoparticles suspension biosynthesized by Aspergillus niger (An),

Aspergillus tubulin (At), Aspergillus fumigatus(Af), Fusarium oxysporum (Fo) and

Penicillium citrinum (Pc) under Transmission Electron Microscope (TEM), the view of

the formed colour due to the formation of ZnONPs (Cv)

Cv

Hefny, et al.

Table 2. Inhibition zone of ZnO (100g/ml) nanoparticles biosynthesized by tested fun against

Gram positive and Gram negative bacterial strains compared to some tested antibiotics

Tested bacteria

Tested fungi

Diameter of inhibition zone (mm)

B. cereu

s

L. m

on

ocyto

gen

es

S. a

ureu

s

P. a

erogin

osa

S. en

terica

E. co

li

Stadard fungi

A. tubingensis Mossery Aumc No. 6915 25±0.23 17±0.14 19±0.11 16±0.10 13±0.11 17±0.25

A. fumigatus Fresenius No. 48 23±0.16 18±0.19 21±0.12 14±0.09 16±0.08 15±0.11

A. terreus Thom Aumc No. 75 24±0.11 23±0.17 22±0.15 11±0.15 13±0.16 13±0.15

Local isolates

A. niger 26±0.21 23±0.11 24±0.15 17±0.18 15±0.26 18±0.26

A. tubulin 25±0.13 22±0.13 25±0.12 14±0.16 18±0.18 15±0.17

A. fumigatus 26±0.16 25±0.18 26±0.19 15±0.17 16±0.13 17±0.18

P. citrinum 14±0.13 12±0.19 15±0.10 12±0.13 10±0.23 12±0.19

F. oxysporum 25±0.15 24±0.17 26±0.14 13±0.22 12±0.27 14±0.16

Control (Antibiotics)

Ofloxacin (5 µg) 27±0.11 29±0.15 30±0.23 25±0.11 10±0.25 24±0.13

Ampicillin 10µg) 20±0.23 21±0.25 23±0.34 7±0.23 11±0.34 14±0.18

Amikacin (30µg) 22±0.32 28±0.16 23±0.18 25±0.16 25±0.23 27±0.15

Time-kill study

In this study, the time kill measurement was

determined by the actual reduction in bacteria

growth curve at 24 hr., of the culturing period

for S. aureus and E. coli. Results from the time–

kill studies are shown in Figs. 3 and 4 which

demonstrates the growth curve of Gram positive

bacteria S. aureus and Gram negative E. coli, S.

aureus and E. coli bacteria were in contact with

0.1 ml of ZnO nanoparticles. The results

exhibited that ZnO nanoparticles have inhibitory

effects on the growth as compared to bacterial

culture without nanoparticles as a control. These

results demonstrated that ZnO nanoparticles

showed inactivation effect against both types of

bacteria from Fig. 3, Gram-negative bacteria is

more resistance to the ZnO nanoparticles

compared to the Gram-positive bacteria. This

can be related to the cell wall-structure, as the

Gram-positive bacteria have one cytoplasmic

membrane with the multilayer of peptidoglycan

polymer, and a thicker cell wall (Fu et al., 2005)

Whereas Gram-negative bacteria wall is

composed of two cell membranes, an outer

membrane and a plasma membrane with a thin

layer of peptidoglycan (Sirelkhatim et al.,

2015). Besides according to Padmavathy et al.

(2008), ZnO nanoparticles have an abrasive

surface texture which influences the

antibacterial mechanism, which in sequence

destroys the bacterial membrane.

Antibiofilm formation

The results of antibiofilm formation by An-ZnO-NPs, At-ZnO-NPs, Af-ZnO-NPs, Pc- ZnO-


NPs and Fo- ZnO-NPs against Gram negative and Gram positive bacteria was assessed by

microtiter plate-based assay, and shown in Table 3.

The effectiveness of inhibition of the microbial

biofilms of S. aureus, L. monocytogenes and B.

cereus compared to S. enterica, E. coli and

P.aerogenosa by An-ZnO-NPs, At-ZnO-NPs,

Af-ZnO-NPs, Pc- ZnO-NPs and Fo- ZnO-NPs

were shown at a concentration of 100 μg/ ml.

The An-ZnO-NPs were more effective to inhibit

the formation of biofilm by all the tested

bacterial strains compared to all the other

treatment. S. aureus was more resistant than all

the tested bacterial strains against all ZnO

nanoparticles. Because, in the process of

staphylococcal biofilm formation, the

accumulation and development of a mature

stage depend mainly on the polysaccharide

intercellular adhesions (PIA) that promote

bacterial accumulation, especially

polysaccharide poly-N-succinyl-β-1-6

glucosamine (PNAG). PNAG biosynthesis is

regulated by enzymes encoded by the ica ADBC

operon (Maira-Litrán et al., 2002). Further

studies should be done on the efficiency of ZnO

nanoparticles against pathogenic bacterium and

fungi such as Methicillin-resistant Staphylococcus

aureus (MRSA) and Trichophyton rubrum

(T. rubrum) including the changes of

morphological of the strains after in contact with

ZnO nanoparticles. This study will contribute to

the understanding of inactivation effect of ZnO

nanoparticles against pathogenic bacteria.

Underline numbers, strong biofilm

formation, normal numbers, moderate biofilm

formation, bold numbers, weak biofilm

formation. Optical density (OD) values. The

mean OD value of negative controls (ODNC) was

0.070. The treatment was considered biofilm

producers when their OD values were three

times greater than the standard deviation of the

mean ODNC. Additionally, the treatments are

showing ability to produce biofilm were

classified as weak (ODNC < OD ≤ 2×ODNC),

moderate (2×ODNC< OD ≤4×ODNC) or strong

(OD >4×ODNC biofilm producers.

Conclusion

ZnO nanoparticles were successfully been

biosynthesized by five different isolates of fungi

(i.e. Aspergillus niger, Aspergillus tubulin,

Aspergillus fumigatus, Penicillium citrinum and

Fusarium oxysporum) as compared to three

standard strains of fungi. UV-vis absorption

analysis showed that the ZnO nanoparticles

exhibited a peak at around 286 to 296 nm. ZnO

nanoparticles exhibited excellent antibacterial

activity against Gram-positive and Gram-

negative bacteria. ZnO nanoparticles showed

Fig. 4. Effect of ZnO nanoparticles on the

growth of S. aureus in TSB at 37°C

Fig. 3. Effect of ZnO nanoparticles on

the growth of E. coli in TSB at

37°C

Control – E. coli Zn=NPs- E. coli

Control – S. aureus Zn=NPs- S. aureus

Hefny, et al.

better antibacterial activity against S.aureus,

L. monocytogenes and B. cereus compared to

S. enterica, E. coli and P. aerogenosa.

Table 3. Antibiofilm formation by Zn-NPs against some pathogenic bacteria

ZnO

nanoparticles

OD570 Biofilm

formation=3×ODNC P. aeroginosa E. coli S. enterica S. aureus

ODNC 0,061 0,069 0,076 0,071 0,280

Positive control 0,426 0,637 0,493 0,465 0,280

An-ZnO-NPs 0,074 0,053 0,058 0,071 0,280

Af-ZnO-NPs 0,076 0,066 0,056 0,304 0,280

At-ZnO-NPs 0,086 0,067 0,073 0,462 0,280

Fo-ZnO-NPs 0,096 0,092 0,103 0,124 0,280

Pc-ZnO-NPs 0,304 0,25 0,22 0,226 0,280

An-ZnO-NPs, A. niger; At-ZnO-NPs, A. tubulin; Af-ZnO-NPs, A. fumigatus; Fo- ZnO-NPs, F. oxysporum and Pc- ZnO-NPs,

P. citrinum

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‏Aspergillus‏هزارع‏فطريح‏لأىاع‏هي‏التخليق‏الحيىي‏لجسيواخ‏الزك‏الاىيح‏تاستخذام‏هرشحاخ

‏سالثح‏لجراماللجرام‏و‏الوىجثحالوضادج‏للثكتيريا‏‏نوخصائصه‏Penicilliumو‏‏Fusariumو

‏هحجىب‏أحوذ‏هرغي‏سوير‏–عثذالفتاح‏تراهين‏إحسي‏‏–الزاهك‏تراهين‏إفاطوح‏‏–‏حفي‏السيذ‏هي

يظش – جبيؼخ انضقبصق –كهخ انضساػخ –ى انكشوثىنىجب انضساػخ قس

وقذسرهب ػه ي انزشثخ رىضح انذساسخ انحبنخ وجىد ػلاقخ إجبثخ ث قذسح رحم يؼذ انضك نهفطشبد انؼضونخ

انز رى ثشكم و سضوسفش ثؼض انجبربد فطشخ ي أىاع 5ورى ػضل (ZnO) أكسذ انضك بى رخهق جضئبد

رز إن رؼشفهب ثبءا ػه انخظبئض انىسفىنىجخ وكبذ هز انؼضلادانششقخ ف يظش ورى يحبفظخطجؼ ف

Aspergillus niger (An), Aspergillus tubulin (At), Aspergillus fumigatus(Af), Penicillium

citrinum (Pc) and Fusarium oxysporum (Fo) زخذيذ هز انؼضلاد ف رخهق جسبد أكسذ انضكاسوقذ

ثبسزخذاو Fo- ZnO-NPs)و Pc- ZnO-NPs و Af-ZnO-NPs و At-ZnO-NPs و (An-ZnO-NPs ، انبىخ

A. tubingensis Mosseray AUMC No.6915, A. fumigatusد انضك يقبسخ إن سلالاد انشاجغ يبكجشز

Fresenius AUMC No.48 and A. terreus Thom AUMC No.75 وجىد ال رى اخزجبسوقذ ZnO-NPs

-UV بىيزش، وانز رظهش ف أطبف 592 إن 585 يهحىظ ػذ ضىئ خلال انكشف ػ رسوح ايزظبص انزكى ي

Vis بى صك وهظهشد زبئج انكشوسكىة الانكزشو انبفز أ جضئبد انأو ،طذي انسطح انجلاصيظبهشح ثسجت

An-ZnO-NPs و At-ZnO-NPs و Af-ZnO-NPs و Pc- ZnO-NPs و Fo- ZnO-NPs كبذ رو رشكت

انبىخ شبطب يزبصا يضبدا انضك جسبدبىيزش( ونقذ اظهشد 033 إن 03م سذاس الاضلاع )كثهىس ػه ش

ضذ شبطب يضبدا شد جضئبد انبىأظه وقذ سبنجخ نجشاو.اننجشاو وانىججخ انجكزشب كلا ي ضذ انخزجشح نهجكزشب

نجشاو بنجكزشب انسبنجخيقبسخ ثـثشكم أفضم و L. monocytogenes و S. aureus انجكزشب انىججخ نجشاو

S. enterica و E. coliو P. aerogenosa. رى رحهم فؼبنخ رثجظ الأغشخ انحىخ انكشوثخ نـ S.aureus

033ثزشكض P.aerogenosa و E. coli و S. enterica يقبسخ ثـ B. cereus و L. monocytogenes و

‏.شاو / يمجيكشو

‏ـــــــــــــــــــــــــــ

‏الوحكوــــــىى:

جبيؼخ ػ شس. –كهخ انضساػخ –أسزبر انكشوثىنىجب انضساػخ ‏خالذ‏عثذالفتاح‏الذجذج‏د.أ.‏-1

جبيؼخ انضقبصق. –كهخ انضساػخ –أسزبر وسئس قسى انكشوثىنىجب انضساػخ ‏هـــىيـــــذا‏هحوذ‏لثية.‏أ.د‏-2

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